Adult stem cells are of considerable biological interest due to their essential role in normal tissue homeostasis as well as their relevance for cancer and other disease processes. The mouse prostate gland can undergo multiple rounds of regression in response to androgen-deprivation followed by androgen-induced regeneration, implying that the prostate epithelium must contain a resident pool of long-term stem cells. Consequently, the mouse prostate should represent an excellent system for studying the function and molecular regulation of adult stem cells, but the properties of these cells are not yet well-defined in vivo. Our preliminary studies indicate that a known regulator of prostate epithelial differentiation, the homeobox gene Nkx3.1, marks an androgen-independent cell (AIN) population that contains prostate epithelial progenitors. In particular, we have employed genetic lineage-marking using inducible Cre-mediated recombination in vivo to demonstrate that AINs can give rise to 2 distinct cell types, indicating that they are bipotential. Furthermore, we have shown that Nkx3.1 mutant mice display progressive prostate epithelial defects in a serial regression/regeneration assay, suggesting that Nkx3.1 is required for stem cell maintenance. Based on our preliminary findings, we hypothesize that the AIN population contains bipotential prostate epithelial progenitors, and raise the possibility that these progenitors may correspond to prostate epithelial stem cells. Consequently, we will now pursue 3 linked specific aims to investigate the molecular basis for multipotent differentiation and self-renewal of prostate epithelial progenitor cells, focusing on the AIN population: 1) Analysis of functional properties of prostate epithelial progenitor cells in vivo by determining the relationship of AINs to label-retaining cells (LRCs), and by investigating their ability to undergo self- renewal. 2) Analysis of mouse epithelial progenitor cells ex vivo by examining the ability of purified cell populations to display cell type-specific differentiation and self-renewal in tissue recombination/grafting assays and clonal cell culture. 3) Investigation of molecular pathways regulating prostate progenitor cell function through analyzing the role of Nkx3.1 in regulating prostate stem cell maintenance, and by identifying regulatory pathways and novel cell-surface markers for prostate epithelial progenitors. Relevance for public health: Our proposed studies of prostate epithelial progenitor cells should have significant implications for the origin of putative "cancer stem cells" during prostate carcinogenesis. These studies may also provide insights into the molecular processes leading to defects in stem cell maintenance with increasing age, as well as the relationship between aging and prostate disease. [unreadable] [unreadable] [unreadable]